Not Applicable
Not applicable.
The present invention relates to cells and non-human transgenic animals that have been engineered to be deficient in the gene encoding the melcanocortin-3 receptor protein (MC-3R). It is shown herein that MC-3R deficient transgenic animals have increased fat mass and reduced lean body mass, showing that the MC-3R protein is involved in the regulation of body fat and lean body mass. The MC-3R deficient transgenic animals of the present invention, including a MC-3R/MC-4R double knockout mouse, can be used to select for and test potential modulators (e.g., agonists or antagonists) of MC-3R, as well as dual modulators of MC-3R and MC-4R. It is shown herein that MC-3R serves a non redundant role, when compared to MC-4R, in the regulation of energy homeostasis. To this end, the present invention also relates to methods of screening for MC-3R modulators which effect body weight and associated methods of treating various disorders or diseases responsive to the action of one or more of the melanocortin receptors, including but not limited to obesity (by reducing appetite, increasing metabolic rate, reducing fat intake or reducing carbohydrate craving), diabetes mellitus (by enhancing glucose tolerance, decreasing insulin resistance), hypertension, hyperlipidemia, osteoarthritis, cancer, gall bladder disease, sleep apnea, depression, anxiety, compulsion, neuroses, insomnia/sleep disorder, substance abuse, pain, male and female sexual dysfunction (including impotence, loss of libido and erectile dysfunction), fever, inflammation, immunomodulation, rheumatoid arthritis, learning memory, modulation of cytokine release, skin tanning, acne and other skin disorders, neuroregeneration and neuroprotective and cognitive and memory enhancement including the treatment of Alzheimer""s disease.
Melanocortin receptors belong to the rhodopsin sub-family of G-protein coupled receptors (GPCR""s). Five different subtypes are known. These melanocortin receptors bind and are activated by peptides such as xcex1-, xcex2, or xcex3-melanocyte stimulating hormones (xcex1-, xcex2-, xcex3-MSH) derived from the pro-opiomelanocortin (POMC) gene. A wide range of physiological functions are believed to be mediated by melanocortin peptides and their receptors.
Desarnaud et al. (1994, Biochem J. 299 (2): 367-372) disclose a cDNA clone encoding mouse MC-3R.
Roselli-Rehfuss et al. (1993, Proc. Natl. Acad. Sci 90: 8856-8860) disclose a cDNA clone encoding rat MC-3R cDNA.
U. S. Pat. No. 5,622,860 (issued Apr. 22, 1997) and U.S. Pat. No. 5,703,220 (issued Dec. 30, 1997) to Yamada and Gantz, disclose DNA molecules which encode human MC-3R and human MC-4R, respectively (see also Gantz, et al., 1993, J. Biol. Chem. 268(11): 8246-8250).
The agouti mouse represents a naturally occurring obese rodent, with a late life onset of obesity which is not corticosterone dependent. The obesity in this model results from the ectopic expression of the 131 amino acid agouti protein. Agouti is normally only expressed in the skin where it controls hair color. The protein is a paracrine antagonist of the melanocortin-1 receptor (MC-1R), a G-protein coupled receptor of the hair follicle. MC-1R agonism, through its natural ligand, xcex1-MSH raises cAMP and the expression of the enzyme tyrosinase. Low levels of tyrosinase, which result from agouti antagonism of MC-1R, result in reduced conversion of the hair color pigment pheomelanin to eumelanin. As a result a light (agouti) rather than black hair color results. The obese phenotype of the agouti mouse was ascribed to the expression of agouti in the brain, where it antagonizes MC-3R and MC-4R receptors. This conclusion was corroborated by the generation of an MC-4R knockout mouse which recapitulates the obese phenotype of the agouti mutant mouse (see U.S. Pat. No. 5,932, 779, issued Aug. 3, 1999 to Lee et al.) In rodents, MC-4R has been implicated as a key regulator of feeding behavior which regulates body weight through studies with peptide agonists and antagonists (Fan et al., 1997, Nature 385:165-168) and with a MC-4R knock-out mouse (Huszar et al., 1997, Cell 88:131-141, see also U.S. Pat. No. 5,932,779, issued Aug. 3, 1999 to Lee et al).
It is desirable to discover new drugs for the treatment of body weight disorders which selectively modulate a melanocortin receptor within the host.
It is also desirable to identify additional receptor targets which are involved in regulating body weight.
The present invention also addresses and meets these needs by disclosing MC-3R-deficient animal cells and/or MC-3R/MC-4R deficient animal cells, related non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are also MC-3R-deficient or MC-3R/MC-4R deficient.
The present invention addresses and meets these needs by disclosing methods of screening for compounds.which effect body weight comprising the screening and selection of compounds which modulate the MC-3R.
The present invention relates to animal cells which are homozygous for an MC-3R deficiency due to a disruption in the gene(s) encoding MC-3R. To this end, the present invention also relates to non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are MC-3R deficient (MC-3R null) due to a disruption in the gene(s) encoding MC-3R.
The present invention further relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are heterozygous for a functional MC-3R gene native to that animal.
The present invention also relates in part to animal cells, non-human transgenic embryos and non-human transgenic littermates having a non-native gene encoding a MC-3R protein expressed either in the presence or absence of the native (wild type) MC-3R. Preferably, the non-native MC-3R gene is the human MC-3R gene.
The present invention also relates to transgenic embryos, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are either homozygous, heterozygous or hemizygous for deletion of at least a portion of the MC-3R gene in combination with a homozygous, heterozygous or hemizygous deletion at separate alleles which in their wild type form encode at least one additional melanocortin receptor, especially a melanocortin receptor shown to be involved in body weight regulation, such as MC-4R. Therefore, aspects of the invention relate to transgenic embryos, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are MC-3Rxe2x88x92/+/MC-4Rxe2x88x92/xe2x88x92; MC-3Rxe2x88x92/+/MC-4R-4xe2x88x92/+; MC-3Rxe2x88x92/xe2x88x92/MC-4Rxe2x88x92/+, as well as hemizygous alternatives in reference to the two separate alleles. An especially preferred aspect of the present invention relates to MC-3Rxe2x88x92/xe2x88x92/MC-4Rxe2x88x92/xe2x88x92 double knockout mice and related transgenic embryos, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates.
The transgenic cells and animals of the present invention are useful in the study of the effect of modulators on the activity of the MC-3R gene and/or protein or the expression of the MC-3R gene and/or protein as concerning the regulation of body weight, including but not limited to disorders such as obesity, diabetes, cardiovascular disease, anorexia, cachexia, cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy.
The present invention also relates to MC-3R-based assays to select for modulators of this receptor protein which affect regulation of body weight through the various known disorders associated with regulation of body weight, as described herein. For example, a MC-3R modulator may be used to treat these body weight disorders, such as a MC-3R agonist to treat obesity or a MC-3R antagonist to treat anorexia and related disorders. These assays may be cell-based assays or may utilize membrane preparations which comprise the MC-3R. Modulation of the MC-3R may also be used to treat growth disorders relating to reduced GH, IGF1 function, treatment of reduced lean body mass as it occurs in the frail elderly, other states that are characterized as resulting from GH deficiency, cancer cachexia, disorders associated with depression and anxiety, obesity (by reducing appetite, increasing metabolic rate, reducing fat intake or reducing carbohydrate craving), diabetes mellitus (by enhancing glucose tolerance, decreasing insulin resistance), hypertension, hyperlipidemia, osteoarthritis, cancer, gall bladder disease, sleep apnea, depression, anxiety, compulsion, neuroses, insomnia/sleep disorder, substance abuse, pain, male and female sexual dysfunction (including impotence, loss of libido and erectile dysfunction), fever, inflammation, immunemodulation, rheumatoid arthritis, learning memory, modulation of cytokine release, skin tanning, acne and other skin disorders, neuroregeneration and neuroprotective and cognitive and memory enhancement including the treatment of Alzheimer""s disease.
As used herein, the term xe2x80x9cfunctionalxe2x80x9d is used to describe a gene or protein that, when present in a cell or in vitro system, performs normally as if in a native or unaltered condition or environment. Therefore, a gene which is not functional (i.e., xe2x80x9cnon-functionalxe2x80x9d, xe2x80x9cdisruptedxe2x80x9d, xe2x80x9calteredxe2x80x9d, or the like) will encode a protein which does not function as a wild type, native or non-altered protein, or encodes no protein at all. Such a non-functional gene, such as a non-functional MC-3R gene, may be the product of a homologous recombination event as described herein, where a non-functional gene is targeted specifically to the region of the target chromosome which contains a functional form of the gene, resulting in a xe2x80x9cknock-outxe2x80x9d of the wild type or native gene.
As used herein, a xe2x80x9cmodulatorxe2x80x9d is a compound that causes a change in the expression or activity of MC-3R, or causes a change in the effect of the interaction of MC-3R with its ligand(s), or other protein(s), such as an agonist or antagonist.
As used herein in reference to transgenic animals of this invention, we refer to xe2x80x9ctransgenesxe2x80x9d and xe2x80x9cgenesxe2x80x9d. As used herein, a transgene is a genetic construct including a gene. The transgene is integrated into one or more chromosomes in the cells in an animal by methods known in the art. Once integrated, the transgene is carried in at least one place in the chromosomes of a transgenic animal. A gene is a nucleotide sequence that encodes a protein, or structural RNA. The gene and/or transgene may also include genetic regulatory elements and/or structural elements known in the art.
As used herein, the term xe2x80x9canimalxe2x80x9d is used herein to include all mammals, except that when referring to transgenic animals, the use of this term excludes humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A xe2x80x9ctransgenic animalxe2x80x9d is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus. This introduced DNA molecule can be integrated within a chromosome, or it can be extra-chromosomally replicating DNA. Unless otherwise noted or understood from the context of the description of an animal, the term xe2x80x9ctransgenic animalxe2x80x9d as used herein refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If offspring in fact possess some or all of the genetic information, then they, too, are transgenic animals. The genetic information is typically provided in the form of a transgene carried by the transgenic animal.
As used herein, a xe2x80x9ctargeted genexe2x80x9d or xe2x80x9cknock outxe2x80x9d (KO) is a DNA sequence introduced into the germline of a non-human animal by way of human intervention, including but not limited to, the methods described herein. The targeted genes of the invention include nucleic acid sequences which are designed to specifically alter cognate endogenous alleles, especially endogenous alleles which encode MC-3R, or alternatively, both MC-3R and MC-4R. The xe2x80x9cknock outxe2x80x9d can be the result of an altered, or preferably, completely deleted MC-3R gene, but also includes but is not limited to MC-3R (and MC-4R) gene deletions, gene modifications and or gene insertions which render the native gene nonfunctional or at least substantially nonfunctional, producing a xe2x80x9cknock outxe2x80x9d transgenic animal, or can lead to a MC-3R (or MC-3R and MC-4R) receptor with altered expression or activity. As noted above, a non-human transgenic animal without an activated MC-3R gene can be used to evaluate the role of MC-3R in obesity and other associated disorders, while a MC-3R/MC-4R knock out can be used to evaluate the role of MC-3R/MC-4R dual modulators in obesity and other disorders described herein.
As used herein, xe2x80x9cMC-1Rxe2x80x9d refers to the melanocortin-1 receptor.
As used herein, xe2x80x9cMC-3Rxe2x80x9d refers to the melanocortin-3 receptor.
As used herein, xe2x80x9cMC-4Rxe2x80x9d refers to the melanocortin-4 receptor.